Protein Kinase C (PKC) is a family of serine-threonine kinases which play an important role in many cellular functions. So far, at least ten different isoforms of PKC have been identified and broadly classified into three major families named classical PKC (cPKC), novel PKC (nPKC) and atypical PKC (aPKC). Of these three families, the classical type consisting of the .alpha., .beta. (I and II), and .gamma. is the best characterized and most abundant. Enzymes in the cPKC family are activated by diacylglycerol in the presence of phosphatidylserine and Ca.sup.++ subsequent to various cell activation mechanisms. However, enzymes in the nPKC family are independent of Ca.sup.++ and the enzymes in the aPKC family are not readily activated by any of the standard second messengers such as phorbol esters and Ca.sup.++. Since different isoforms of PKC are widely distributed in various tissues and regulate different intracellular functions, estimation of PKC activity is an important step in signal transduction research.
Normally, PKC is assayed by measuring incorporation of .sup.32 P from (.gamma.-.sup.32 P)ATP into suitable substrates, usually histone (type III-SS), in a reaction mixture that contains magnesium, (.gamma.-.sup.32 P)ATP, diacylglycerol, L-.alpha.-phosphatidyl L-serine and Ca.sup.++. In most assays, the PKC reaction is run at 37.degree. C. and at the end of reaction phosphorylated (.sup.32 P) substrate is removed from the rest of the reaction mixture either by using precipitation methods or by using acidic phosphocellulose papers that bind only the basic phosphorylated histone while anionic (.gamma.-.sup.32 P)ATP may be washed away. Both of these methods involve (.gamma.-.sup.32 P)ATP and relatively expensive radioactive measurement techniques.
A procedure has been developed in which the PKC activity is measurable using non-radiolabeled ATP and a 96-well micro-plate. Our method uses a simple substrate immobilization technique and a colorimetric analysis to determine the extent of substrate phosphorylation. The method is simple, sensitive, rapid, and specific for PKC. This method may also be used for various other protein kinases by immobilizing suitable substrates and adjusting the incubation conditions.
The second type of enzymes that can be assayed by this invention, protein phosphatases, are regulatory enzymes that antagonize the action of protein kinases within the cell. Protein phosphatases fall into two general categories: those with specificity for phosphoserine or phosphothreonine residues and those with specificity for phosphotyrosine residues. Protein tyrosine phosphatases (PTPases) play a critical role in lymphocyte signaling, cell cycling, bacterial virulence and tumorigenesis. The serine/threonine class of protein phosphatases are also involved in many aspects of cellular regulation including immunosuppression, shellfish poisoning and cell cycle control. The serine/threonine-specific protein phosphatases are divided into two groups, type-1 and type-2. The type-1 protein phosphatases (PP-1) preferentially dephosphorylate the .beta. subunit of phosphorylase kinase. Type-2 protein phosphatases termed PP-2A, PP-2B and PP-2C dephosphorylate the .alpha. subunit of phosphorylase kinase. Protein phosphatases activities are normally measured using radiolabeled substrates or by using anti-phosphotyrosine antibodies to estimate the level of dephosphorylation from the specific substrates. The above mentioned techniques involve costly equipment or hazardous radioisotopes. A need exists for a simple, non-radioactive assay for estimating protein phosphatases. The present invention is a procedure in which the PP-1 activity is measured using non-radiolabeled substrate (phosphorylase-b) and a 96-well ELISA plate reader. The method uses a simple substrate immobilization technique and a colorimetric analysis to determine the extent of dephosphorylation. The method is simple, sensitive, rapid and specific for pp-1. This method may also be used for various other protein phosphatases by immobilizing suitable substrates and adjusting the incubation conditions.